Behavioral and cognitive disorders caused by genetic lesions, environmental insults or age related dementia are serious societal problems, contributing to a loss of quality of life for affected individuals and their families. The incidence of intellectual disability in the United States is 1-2%, costing tens of billions of dollars/year in health care and lost productivity. A key to defining therapeutic strategies that can ameliorate these disorders is a fundamental understanding of the cellular and molecular mechanisms regulating how neuronal circuits encode, process, and retain information. Synapses are the basic components of information storage and plasticity in our brains. Over the last decade, we have used molecular, cellular and reverse genetic approaches to identify and characterize proteins involved in the assembly, function and plasticity of vertebrate synapses. Increasingly, our data has shown that many of these proteins are transcribed from large multi-gene families spanning hundreds of kb of genomic DNA and comprised of multiple alternatively spliced exons. This complexity, as well as the cost and time associated with the generation of conditional knockout or knockin mice, has severely hampered progress in the field. What is needed is a simple, cost-effective strategy, akin to transgenics, for creating mice deficient in individual or combinations of proteins. Recent advances in transgenic technology using lentiviruses combined with interference RNAs are well poised to meet this challenge. Over the last five years, we have developed isoform specific short-hairpin RNAs (shRNAs) against numerous synaptic proteins as well as a collection of lentiviral vectors expressing XFP-tagged reporter proteins for the cell-autonomous and synapse specific analysis of pre and postsynaptic function. In the present application, we propose to integrate these technologies and the CRE/lox system to create an innovative set of lentiviruses capable of conditionally inactivating and expressing multiple neuronal proteins. Given their importance in the assembly and plasticity of synapses, we propose to use shRNAs against the structurally related presynaptic active zone proteins Piccolo and Bassoon to evaluate this new strategy. These conditional knockdown mice for Piccolo and/or Bassoon will be invaluable for assessing the shared and unique functions of these proteins during neuronal differentiation, axonal pathfinding, synapse formation, and in mechanisms of presynaptic plasticity at vertebrate synapses. PUBLIC HEALTH RELEVANCE: This grant application describes experiments designed to create a set of conditional transgenic knockdown mice deficient in the expression of the structurally related presynaptic active zone proteins Piccolo and Bassoon. In Aim 1, we propose to design, build and test the next generation of lentiviral vectors for the cost effective creation of conditional knockdown mice. Specifically, we will create a virus expressing three mini-genes: one an shRNA for Piccolo knockdown, the second a YFP-tagged Synapsin1a for labeling presynaptic boutons, and the third a red fluorescent protein variant (mCherry) for selecting mice with the integrated transgenes. The expression of each will be placed under the control of the CRE/lox system. The functionality of these vectors will be tested in HEK293 cells and cultured hippocampal neurons. In Aim 2, we will create and characterize a transgenic mouse using the lentiviral vector created in Aim1. In Aim 3, we will expand this technology to create a lentiviral vector capable of knocking down two or more synaptic proteins by expressing shRNAs for each protein under separate polymerase III promoters. This technology will be invaluable for studies of individual or families of proteins thought to perform similar functions. Moreover, it is ideally suited for a molecular replacement strategy that simultaneously eliminates the expression of one synaptic protein while replacing it with a mutated or altered version. Such a strategy could become crucial for the generation of mouse models of specific psychiatric, neurodegeneartive or neurodevelopment disorders such as depression, schizophrenia, or Autism Spectrum Disorders, or Alzheimer's disease.